Interim Hygiene Protocols for Amphibian field staff and

STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
ECOSYSTEMS BRANCH, MINISTRY OF ENVIRONMENT, BRITISH COLUMBIA
Interim Hygiene Protocols for Amphibian field staff and researchers
PURPOSE
To describe hygiene protocols to be used by amphibian field staff and researchers to
reduce risk of disease transmission among sites and among animals within a site.
DESCRIPTION
Over the past few decades emerging infectious diseases such as the amphibian chytrid
fungus (Batrachochytrium dendrobatidis) and ranavirus have been implicated in the
decline of amphibian populations from many sites around the world. It is thought that
field staff could act as potential vectors of transmission introducing these diseases into
new sites and to naïve species. The risk of this mode of transmission is not fully
understood compared to other modes of transmission, for example by vectors such as
waterfowl and other wildlife. However, to reduce the risk, it is essential that all field
workers incorporate the hygiene protocol into their research plans as Standard Operating
Procedure (SOP).
To encourage compliance, the SOP below aims to strike a balance between reducing
risk of disease transmission and increasing ease of protocol implementation under field
conditions.
The protocols below address two levels of disease transmission: 1) the risk of disease
transmission between sites; and 2) the risk of disease transmission among individuals
within a site.
RESPONSIBILITY
This protocol applies to all field staff working in aquatic habitats sampling
amphibians in freshwater lentic and lotic wetlands.
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
MATERIALS
• Plastic tote or box to store field equipment.
• Buckets and totes for mixing bleach solution and soaking equipment
• Spray bottle for bleach solution
• Scraper, brushes, hand-brushes
• Rubber dishwashing gloves, rubber apron
• Clean plastic containers and bags for temporarily holding animals
• Household bleach (contains active ingredient 5-6.5% sodium hypochlorite solution)
• Vinyl or nitrile gloves for handling amphibians
• Sterilizing solutions (ethanol) and alcohol burner/lighter for disinfecting instruments
if toe-clipping or marking animals.
• Container for waste disposal
STANDARD OPERATING PROCEDURES (SOP)
I. SOP when moving between sites
Definition of site
At geographic scales of tens of kilometres, watersheds and major geographical
barriers should be used to designate separate sites.
Each tributary of a river should be considered a separate site. Wetlands, ponds and
lakes separated by dry land should be considered separate sites.
Site designation is particularly difficult at smaller geographic scales and with small
isolated water bodies. At scales less than 500 m, if the water bodies remain separate
under high water/flood conditions then they should be considered separate sites. Within a
stream (at distances less than 500 m) sampling should occur in a downstream direction
where possible. Each stream and each upstream location should be considered separate
sites.
Equipment treatment
Equipment such as wetsuits, waders, footwear, nets, buckets, and traps may act as
vectors of disease spread. In this SOP we recommend household bleach for disinfection
because it is widely available and it is easy to dispose under field conditions if the
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
precautions below are followed. However, there are a number of other disinfectants that
have been tested (Table 1) that offer a variety of other advantages. The SOP can be used
with these chemical disinfectants instead of bleach, but manufacturer’s recommendations
and Material Safety Data Sheet requirements for use and disposal must be followed. It is
important to ensure that disinfectants do not leave a residue on equipment, as the residue
can be harmful to amphibians
1. Before leaving a site, field workers must scrub using a hand brush and rinse using the
pond/stream water to remove mud, algae, plants, snails and other invertebrates from
all equipment. Disinfection procedures work best on cleaned equipment, free of
debris.
2. A bleach solution with 0.2 % sodium hypochlorite and exposure time of 10 minutes
has been shown to be effective against Bd (Johnson et al 2003). Commercial
household bleach sold in North America often contains 6.15% sodium hypochlorite,
but concentrations can vary. To prepare the disinfectant solution, add 32 ml of
household bleach to 1 litre of water. This translates to approximately 3.5 cups (0.85
litre) of bleach to one tall bucket or tote (~ 25 litres) of water. In the absence of
municipal/well water supply, water from the pond or stream can be used.
3. All equipment must be soaked in the bleach solution for a minimum of 15 minutes.
Small items such as dipnets, and sample containers can be immersed in the bleach
solution in a bucket or plastic tote. Larger items such as chest waders, paddles, boats,
canoes, meter sticks and other survey equipment should be thoroughly soaked with
the bleach solution using a spray bottle.
4. The bleach solution can be rinsed off after 15 minutes with clean water from a well or
municipal supply. However, if clean treated water is not available, the items can be
hung out to dry, preferably in sunlight, so that the bleach evaporates completely from
the equipment.
5. The bleach solution can damage exposed skin and clothing. Dishwashing gloves and
rubber aprons should be worn to protect clothing and skin from exposure to the
bleach solution.
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
6. The SOP is most easily carried out back at the laboratory or field station. All
equipment should be stored in a waterproof box or tote during transportation to
prevent contaminating the vehicle and preventing the vehicles from acting as
secondary sources of cross contamination.
7. If the equipment needs to be used immediately at another site prior to returning to the
laboratory or field station, the SOP should be carried out on a road or other
impermeable surface away from the waterbody, so that no bleach contaminated water
enters surface water bodies. If time permits, all equipment should be dried
completely between sites. This is facilitated by having two sets of gear, one of which
is drying while the other is in use. If the equipment needs to be used immediately at
another site, residual bleach from nets and other equipment should be rinsed off using
water from the second site, again working away from the waterbody. Even trace
amounts of residual bleach can adversely affect amphibians on contact.
8. In the field, the bleach solution is best disposed far from the waterbody by pouring
over an asphalt, hard roadbed or concrete surface where it breaks down in sunlight
and evaporates. Ensure no bleach water enters surface waters directly.
9. Washing road vehicles at a carwash between watersheds is desirable. All off-road
vehicles, boats, canoes, and other floatation devices should be subject to the same
SOP as sampling equipment. Large equipment, such as boats and seine nets, are most
easily handled by spraying with or soaking in the bleach solution and then rinsing off
at a carwash using high pressure hot water rinses.
Special Sites
The above SOP should be sufficient to reduce the risk of disease transmission under
most circumstances. However, at sites where there have been known disease outbreaks or
sites with highly endangered amphibian populations extra precautions are necessary. The
simplest solution would be to have dedicated field gear that is used only at that site.
II. SOP under Special Circumstances
The above SOP should be implemented under all normal operating conditions.
However, if for unforeseen and unplanned reasons it is not possible to adhere strictly to
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
the above SOP the following precautions can be taken to reduce the risk of disease
transmission.
1. At a minimum, all equipment should be scrubbed and rinsed thoroughly to remove
debris, algae, invertebrates and mud.
2. Complete drying between sites can reduce the risk of transmission of chytrid fungus
(Bd) but is not effective against ranavirus.
3. If it is possible to heat water, equipment should be soaked in water > 60° C for 30
minutes. This method may be practical for small equipment such as dipnets that come
in direct contact with the animals.
III. SOP for handling amphibians within a site
It is important to assume that not all animals from a site are infected and that handling
multiple animals without adequate caution will significantly increase the risk of between
animal disease transmissions. The following SOP should be followed to ensure that
handling by field staff does not increase the risk of an animal being exposed to a
pathogen.
1. All field staff must ensure that their hands are well cleaned and free of residual
chemicals such as sunscreen and insecticides at the start of the survey.
2. All field staff must wear disposable gloves when capturing amphibians. Nonpowdered
vinyl and nitrile gloves are preferred, as latex has been shown to have
toxicity towards frog embryos and tadpoles.
3. Gloves should be changed between animal captures. If under unforeseeable
circumstances access to gloves is limited, it is better to capture animals with bare
hands and wash hands thoroughly in water to which the animals are habitually
exposed, rather than to use the same pair of gloves to handle multiple animals.
4. Captured animals must be housed in separate disinfected containers or disposable
bags such as plastic Ziploc bags.
5. The duration of restraint should be as short as possible. The greatest stress and risk of
disease transmission between animals is during restraint. Every effort should be made
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
to process animals at the site of capture and avoid captivity. Containers used to house
individuals should not be reused unless the containers have been treated using 4%
bleach solution as described in SOP #1 above.
6. Fresh disposable gloves must be worn when measuring, swabbing or marking the
animal.
7. Surgical instruments such as scissors used for toe clipping should be dipped in 70%
ethanol and flamed briefly between uses. A bic lighter is useful and effective for
flaming surgical instruments in the field. Wiping down with ethanol alone kills the
pathogens but does not completely destroy the pathogen DNA. This may increase the
number of false positives if the animals are being sampled for disease prevalence.
8. Instruments used for inserting PIT tags and other tags should also be sterilized
between animals. Fresh disposable syringes should be used for each animal.
9. The tuberculin syringes used for fluorescent elastomer paint injections present a
special problem. The needles can neither be changed between animals nor is it
practical to use a fresh syringe for each marked animal. At this time the
recommendation is that the needles should be sterilized with appropriate
viricide/bactericide (or dipped in 70% ethanol) and rinsed with distilled water
between making animals. The efficacy of this method in preventing between animal
disease transmission needs to be assessed.
FURTHER INFORMATION
When in doubt, field staff should use the precautionary principle and use the highest
standards of cleanliness possible. An additional half hour spent following the most
stringent SOP is well worth the effort. A number of other protocols, using a variety of
disinfection agents are listed below.
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
RELEVANT LITERATURE
Daszak P, Cunningham AA, Hyatt AD. 2001. Draft guidelines for international
translocation of amphibians with respect to infectious diseases. Attachment 6. In:
Speare R and Steering Committee of Getting the Jump on Amphibian Disease.
Developing management strategies to control amphibian diseases: Decreasing the
risks due to communicable diseases. School of Public Health and Tropical Medicine,
James Cook University: Townsville. 2001: 150-156.
Department of Environment and Heritiage. 2006. Threat abatement plan for infection of
amphibians with chytrid fungus resulting in chytridiomycosis. Department of
Environment and Heritage, Commonwealth of Australia.
http://www.deh.gov.au/biodiversity/threatened/publications/tap/chytrid/
Gutleb, A. C., M. Bronkhorst, J. H. J. van den berg, and A. J. Murk. 2001. Latex
laboratory gloves: an unexpected pitfall in amphibian toxicity assays with
tadpoles. Environmental Toxicology and Chemistry 10:119-121.
Johnson M, Berger L, Philips L, Speare R. 2003. Fungicidal effects of chemical
disinfectants, UV light, dessication and heat on the amphibian chytrid,
Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 57:255-260.
Speare, R., L. Berger, L. F. Skerratt, R. A. Alford, D. Mendez, S. Cashins, N. Kenyon, K.
Hauselberger, and J. J. L. Rowley. 2004. Hygiene protocol for handling amphibians
in field studies. Amphibian Disease Group, James Cook University, Townsville 4811,
Australia. http://www.jcu.edu.au/school/phtm/PHTM/frogs/field-hygiene.pdf
US Forest Service Intermountain Region. Technical Chemical Information for
Disinfecting Aquatic Invasive Species (excellent resource for calculating
disinfectant dilutions)
http://www.fs.fed.us/r4/resources/aquatic/guidelines/chemical_tech_final.xls
Webb, R., D. Mendez, L. Berger, and R. Speare. 2007. Additional disinfectants effective
against the amphibian chytrid fungus Batrachochytrium dendrobatidis. Diseases
of Aquatic Organisms 74:13-16.
Wellington, R. and R. Haering. 2001. Hygiene protocol for the control of diseases in
frogs. New South Wales National Parks and Wildlife Service Threatened Species
Management Information Circular No 6, Hurstville, Australia.
www.npws.gov.au/wildlife/licence/frog.html
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STANDARD OPERATING PROCEDURES: HYGIENE PROTOCOLS FOR AMPHIBIAN FIELDWORK, 2008
Table 1: Summary of disinfectant concentrations and exposure times found to be
effective against Bd. Table reproduced with modification from Speare et al (2004) with
additional information from Johnson et al (2003) and Webb et al (2007).
DISINFECTANT CONCENTRATION TIME REFERENCE
Disinfecting surgical equipment and scales
Ethanol 70% 1 min Speare et al 2004
Webb et al 2007
Virkon 1mg/ml 1 min Speare et al 2004
Benzalkonium
chloride
1 mg/ml 1 min Speare et al 2004
Disinfecting collection equipment, containers, footwear, waders, boats, nets and
other field gear
Household bleach
(sodium
hypochlorite 4% to
6%)
Didecyl dimethyl
ammonium chloride
Quaternary
ammonium
compound 128
4% to 1%
(0.2 to 0.01%
sodium
hypochlorite)
10 min to 30
sec
Speare et al 2004
Johnson et al 2003
Webb et al 2007
1 to 1000 dilution 30 sec Speare et al 2004
Full strength to
1x10 -3
5 mins to 30
sec
Johnson et al 2003
Virkon 1 mg/ml
5 min, 20 sec Johnson et al 2003
2 g/litre
1 min Webb et al 2007
F10 Super
Concentrate
Disinfectant
0.7 ml/litre 1 min Webb et al 2007
TriGene Viricidal
Surface Disinfectant
Cleaner
0.2 ml/litre 1 min Webb et al 2007
DDAC 2 ml/litre 1 min Webb et al 2007
Sodium chloride 10% 5 min, 2 min Johnson et al 2003
Potassium
permanganate
2% 10 min, 5 min Johnson et al 2003
Hot wash for cloth
bags and clothing
60°C or greater 15 mins Speare et al 2004
Heat 60°C 30 minutes Speare et al 2004
Webb et al 2007
Complete drying Effective against Bd 3 hrs or greater Speare et al 2004
(footwear only) but not ranavirus
Sterilising UV light
(1000 mW m -2 Effective against 1 min Speare et al 2004
,
wavelength 254 nm)
ranavirus but not Bd
Johnson et al 2003
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